The present invention relates to a DNA sequence containing a DNA segment coding for a human vasoconstrictive peptide, namely endothelin-2, a precursor protein (or a precursor polypeptide) and a mature protein (or a mature polypeptide) of endothelin-2 and a method for preparing the precursor protein and the mature protein (endothelin-2), and further to a DNA sequence containing a DNA segment coding for human endothelin-3, a precursor protein of human endothelin-3 and a method for preparing endothelin-3.
In this specification, the term "precursor protein" is preferably used to describe a protein which includes an amino acid sequence of a mature peptide and has a portion or all of an amino acid sequence coded with a DNA segment of the peptide at the N-terminus, the C-terminus or both termini thereof.
There have been reports of endothelium-dependent vasoconstrictor reactions to various mechanical and chemical stimuli as well as endothelium-dependent vasodilative reactions. For example, it is known that vasoconstriction can be induced by mechanical loads such as vascular stretch and increased vascular inner pressure, or can be chemically induced by such agents as thrombin. Further, vasoconstriction can be induced by conditions of anoxia. Noradrenaline-induced vasoconstriction can be enhanced by use of neuropeptide Y [K. Takemoto, Proc. Natl. Acad. Sci. U.S.A. 79, 5485 (1982); C. Minth et al., ibid. 81, 4577 (1984)]. Endothelial cell-derived coronary vascular constrictor factors (each having molecular weights of 8,500 and 3,000) are described in K. A. Hickey et al., Am. J. Physiol. 248, C550(1985); and in R. F. O'Brien, J. Cell Physiol. 132, 263 (1987). However, their structures are unknown. An endothelial cell-derived peptide-like substance is also described in M. N. Gillespie et al., J. Pharmac. Exp. Ther. 236, 339 (1985). However, the structure of that substance is also unknown.
Vasopressin is known as a peptide having a vasoconstrictor activity, and the amino acid sequence thereof was determined. There have been no reports, however, that vasopressin was obtained from mammalian or bird vascular endothelial cells. Although there is a report that an angiotensin having a vasoconstrictor activity was obtained from the endothelial cells of bovine aortas [I. Kifor and V. J. Dzav, Circ. Res. 60, 422 (1987)], the angiotensin is a peptide having a molecular weight of only about 1,000.
Some of the present inventors have previously succeeded in isolating porcine endothelin as a peptide having a similar vasoconstrictor activity from the endothelial cells of porcine aortas (Japanese Unexamined Patent Publication No. 206997/1989). Some of the present inventors have also succeeded in isolating human endothelin and cloning porcine endothelin cDNA and human endothelin cDNA (Japanese patent application Nos. 275613/1987, 313155/1987, 148158/1988 and 274454/1988). The mature polypeptides of the porcine endothelin and the human endothelin have the same amino acid sequence, and are referred to as endothelin-1.
Further, the present inventors have filed patent applications with respect to the isolation of rat endothelin and the cloning of its cDNA (Japanese patent application Nos. 174935/1988 and 188083/1988), and this rat endothelin is referred to as endothelin-3.
Furthermore, the present inventors have also filed a patent application with respect to the isolation of mouse endothelin and the cloning of its cDNA (Japanese patent application No. 223389/1988), and this mouse endothelin is referred to as endothelin B.
The amino acid sequences of the endothelin-1, endothelin B and endothelin-3 are shown in FIG. 3 in comparison to one another.
Endothelin is a general term for peptides having a molecular weight of 2500+300 and having 21 amino acid residues, including four cysteine groups located at the lst, 3rd, 11th and 15th residues from the N-terminus of the amino acid sequence, which form two sets of disulfide bonds. One of the combinations of the disulfide bonds may be 1-15 and 3-11 cysteine groups, and the other may be 1-11 and 3-15. The former is higher in ratio of formation and in activity than the latter.
These above-described endothelins have been called variously, and in the present invention, newly unified names for the endothelins are employed in the present invention. They are shown as compared with the previous names as follows:
______________________________________ Newly unified names Previous names ______________________________________ endothelin-1 endothelin A human endothelin porcine endothelin endothelin .alpha. endothelin-B mouse endothelin endothelin B endothelin .beta. endothelin-3 endothelin C endothelin .gamma. rat endothelin ______________________________________
As described above, homologous endothelin peptides have been discovered from various animals. However, no novel hologous genes have been discovered from the same animal species. It is therefore a current subject that novel homologous endothelin is further screened, and the structure and activity of the endothelin is studied, thereby examining its usefulness, and that the novel peptide is cloned by gene recombination to pioneer mass production thereof.